The objective of this project is to characterize the functional role of protein O- GlcNAcylation in crucial areas of stem cell biology. O-GlcNAcylation is a post- translational modification in which a single monosaccharide (O-GlcNAc) is installed on Ser or Thr residues of cytosolic and nuclear proteins. This modification has been found on a wide array of proteins, including transcription factors. Although much is known about the crucial role of O-GlcNAcylation in cellular processes, this modification has yet to be studied in the context of regenerative medicine. I propose herein to characterize the functional role of O- GlcNAcylation in two critical areas of stem cell biology: neuronal differentiation (Aim 1), and the repression of genomic transcripts by polycomb group proteins (Aim 2). In Aim 1, I propose to use Metabolic Oligosaccharide Engineering (MOE) and glycoproteomic analysis to identify the protein substrates of O-GlcNAcylation present during various stages of neuronal differentiation. These studies will aid in the elucidation of the molecular determinants of stem cell fate decisions during neuronal differentiation. In Aim 2 I will study the putative role of O-GlcNAcylation in the maintenance of hESC and adult stem cells by polycomb group proteins (PcGs). Recent work in Drosophila has shown that O-GlcNAcylation governs PcG-mediated gene silencing during development. Herein we will identify both O-GlcNAcylated proteins and the genes that are being modulated by them by performing a chromatin immunoprecipitation (ChIP)-type assay facilitated by MOE. This work may expand the current understanding of the molecular mechanisms underlying self-renewal and pluripotency of hESCs. In summary, the successful completion of the studies proposed herein will constitute the first-ever comprehensive study of protein O-GlcNAcylation in hESCs, and will greatly expand the current understanding of molecular mechanisms governing neuronal differentiation, pluripotency and transcriptional control in hESC biology. PUBLIC HEALTH RELEVANCE: Human embryonic stem cells can be changed into virtually any cell type in the adult body, and thus, have the potential to cure a vast majority of existing human disorders. The results of the studies proposed in this project may lead to a greatly increased understanding of how stem cells retain their ability to be changed into other cell types, and also how the fate of stem cells is decided upon differentiation. Both are critical areas that need to be explored to enable modern regenerative medicine to realize its full potential as tool for the treatment of human diseases, such as Alzheimer's, Parkinson, and Batten disease.